RACK1 regulates specific functions of Gbetagamma

We showed previously that Gbetagamma interacts with Receptor for Activated C Kinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with Galpha subunits or heterotrimeric G proteins but binds free Gbetagamma subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with Gbetagamma promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to Gbetagamma results in inhibition of Gbetagamma-mediated activation of phospholipase C beta2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of Gbetagamma, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the Galpha subunits of G(i), G(s), or G(q). Collectively, these findings suggest a role of RACK1 in regulating specific functions of Gbetagamma.     

Regulator of G-protein signaling 3 (RGS3) inhibits Gbeta1gamma 2-induced inositol phosphate production, mitogen-activated protein kinase activation, and Akt activation

Regulator of G-protein signaling 3 (RGS3) enhances the intrinsic rate at which Galpha(i) and Galpha(q) hydrolyze GTP to GDP, thereby limiting the duration in which GTP-Galpha(i) and GTP-Galpha(q) can activate effectors. Since GDP-Galpha subunits rapidly combine with free Gbetagamma subunits to reform inactive heterotrimeric G-proteins, RGS3 and other RGS proteins may also reduce the amount of Gbetagamma subunits available for effector interactions. Although RGS6, RGS7, and RGS11 bind Gbeta(5) in the absence of a Ggamma subunit, RGS proteins are not known to directly influence Gbetagamma signaling. Here we show that RGS3 binds Gbeta(1)gamma(2) subunits and limits their ability to trigger the production of inositol phosphates and the activation of Akt and mitogen-activated protein kinase. Co-expression of RGS3 with Gbeta(1)gamma(2) inhibits Gbeta(1)gamma(2)-induced inositol phosphate production and Akt activation in COS-7 cells and mitogen-activated protein kinase activation in HEK 293 cells. The inhibition of Gbeta(1)gamma(2) signaling does not require an intact RGS domain but depends upon two regions in RGS3 located between acids 313 and 390 and between 391 and 458. Several other RGS proteins do not affect Gbeta(1)gamma(2) signaling in these assays. Consistent with the in vivo results, RGS3 inhibits Gbetagamma-mediated activation of phospholipase Cbeta in vitro. Thus, RGS3 may limit Gbetagamma signaling not only by virtue of its GTPase-activating protein activity for Galpha subunits, but also by directly interfering with the activation of effectors.     

GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Galpha and Gbetagamma subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Galpha and Gbetagamma) following agonist-induced GPCR (alpha(2A)-adrenergic receptor; alpha(2A)-AR) activation in a cell-free assay system. alpha(2A)-AR membranes were reconstituted with the G-proteins (+/-hexahistidine-tagged) Galpha(i1) and Gbeta1gamma2 and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [35S]GTPgammaS. In the presence of Ni(2+)-coated agarose beads, the activated his-tagged Galpha(i1)his-[35S]GTPgammaS complex was captured on the Ni(2+)-presenting surface. When his-tagged Gbeta1gamma2 (Gbeta1gamma2his) was used with Galpha(i1), the [35S]GTPgammaS-bound Galpha(i1) was not present on the Ni(2+)-coated beads, but rather, it was separated from the beta1gamma2(his)-beads, demonstrating receptor-induced dissociation of Galpha and Gbetagamma subunits. Treatment of the reconstituted alpha(2A)-AR membranes containing Gbeta1gamma2his:Galpha(i1) with imidazole confirmed the specificity for the Ni2+:G-protein surface dissociation of Galpha(i1) from Gbeta1gamma2his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Structure of the parathyroid hormone receptor C terminus bound to the G-protein dimer Gbeta1gamma2

A critical role of the Gbetagamma dimer in heterotrimeric G-protein signaling is to facilitate the engagement and activation of the Galpha subunit by cell-surface G-protein-coupled receptors. However, high-resolution structural information of the connectivity between receptor and the Gbetagamma dimer has not previously been available. Here, we describe the structural determinants of Gbeta1gamma2 in complex with a C-terminal region of the parathyroid hormone receptor-1 (PTH1R) as obtained by X-ray crystallography. The structure reveals that several critical residues within PTH1R contact only Gbeta residues located within the outer edge of WD1- and WD7-repeat segments of the Gbeta toroid structure. These regions encompass a predicted membrane-facing region of Gbeta thought to be oriented in a fashion that is accessible to the membrane-spanning receptor. Mutation of key receptor contact residues on Gbeta1 leads to a selective loss of function in receptor/heterotrimer coupling while preserving Gbeta1gamma2 activation of the effector phospholipase-C beta.     

Gbetagamma subunit combinations differentially modulate receptor and effector coupling in vivo

In vitro, little specificity is seen for modulation of effectors by different combinations of Gbetagamma subunits from heterotrimeric G proteins. Here, we demonstrate that the coupling of specific combinations of Gbetagamma subunits to different receptors leads to a differential ability to modulate effectors in vivo. We have shown that the beta(1)AR and beta(2)AR can activate homomultimers of the human inwardly rectifying potassium channel Kir 3.2 when coexpressed in Xenopus oocytes, and that this requires a functional mammalian Gs heterotrimer. Modulation was independent of cAMP production, suggesting a membrane-delimited mechanism. To analyze further the importance of different Gbetagamma combinations, we have tested the facilitation of Kir 3.2 activation by betaAR mediated by different Gbetagamma subunits. The subunits tested were Gbeta(1,5) and Ggamma(1,2,7,11). These experiments demonstrated significant variation between the ability of the Gbetagamma combinations to activate the channels after receptor stimulation. This was in marked contrast to the situation in vitro where little specificity for binding of a Kir 3.1 C-terminal GST fusion protein by different Gbetagamma combinations was detected. More importantly, neither receptor, although homologous both structurally and functionally, shared the same preference for Gbetagamma subunits. In the presence of beta(1)AR, Gbeta(5)gamma(1) and Gbeta(5)gamma(11) activated Kir 3.2 to the greatest extent, while for the beta(2)AR, Gbeta(1)gamma(7), Gbeta(1)gamma(11,) and Gbeta(5)gamma(2) produced the greatest responses. Interestingly, no preference was seen in the ability of different Gbetagamma subunits to facilitate receptor-stimulated GTPase activity of the Gsalpha. These results suggest that it is not the receptor/G protein alpha subunit interaction or the Gbetagamma/effector interaction that is altered by Gbetagamma, but rather that the ability of the receptor to interact productively with the Gbetagamma subunit directly and/or the G protein/effector complex is dependent on the specific G protein heterotrimer associated with the receptor.     

Gib2, a novel Gbeta-like/RACK1 homolog, functions as a Gbeta subunit in cAMP signaling and is essential in Cryptococcus neoformans

Canonical G proteins are heterotrimeric, consisting of alpha, beta, and gamma subunits. Despite multiple Galpha subunits functioning in fungi, only a single Gbeta subunit per species has been identified, suggesting that non-conventional G protein signaling exists in this diverse group of eukaryotic organisms. Using the Galpha subunit Gpa1 that functions in cAMP signaling as bait in a two-hybrid screen, we have identified a novel Gbeta-like/RACK1 protein homolog, Gib2, from the human pathogenic fungus Cryptococcus neoformans. Gib2 contains a seven WD-40 repeat motif and is predicted to form a seven-bladed beta propeller structure characteristic of beta transducins. Gib2 is also shown to interact, respectively, with two Ggamma subunit homologs, Gpg1 and Gpg2, similar to the conventional Gbeta subunit Gpb1. In contrast to Gpb1 whose overexpression promotes mating response, overproduction of Gib2 suppresses defects of gpa1 mutation in both melanization and capsule formation, the phenotypes regulated by cAMP signaling and associated with virulence. Furthermore, depletion of Gib2 by antisense suppression results in a severe growth defect, suggesting that Gib2 is essential. Finally, Gib2 is shown to also physically interact with a downstream target of Gpa1-cAMP signaling, Smg1, and the protein kinase C homolog Pkc1, indicating that Gib2 is also a multifunctional RACK1-like protein.     

The RACK1 ortholog Asc1 functions as a G-protein beta subunit coupled to glucose responsiveness in yeast

According to the prevailing paradigm, G-proteins are composed of three subunits, an alpha subunit with GTPase activity and a tightly associated betagamma subunit complex. In the yeast Saccharomyces cerevisiae there are two known Galpha proteins (Gpa1 and Gpa2) but only one Gbetagamma, which binds only to Gpa1. Here we show that the yeast ortholog of RACK1 (receptor for activated protein kinase C1) Asc1 functions as the Gbeta for Gpa2. As with other known Gbeta proteins, Asc1 has a 7-WD domain structure, interacts directly with the Galpha in a guanine nucleotide-dependent manner, and inhibits Galpha guanine nucleotide exchange activity. In addition, Asc1 binds to the effector enzyme adenylyl cyclase (Cyr1), and diminishes the production of cAMP in response to glucose stimulation. Thus, whereas Gpa2 promotes glucose signaling through elevated production of cAMP, Asc1 has opposing effects on these same processes. Our findings reveal the existence of an unusual Gbeta subunit, one having multiple functions within the cell in addition to serving as a signal transducer for cell surface receptors and intracellular effectors.     

Gbeta residues that do not interact with Galpha underlie agonist-independent activity of K+ channels.

Gbetagamma subunits interact directly and activate G protein-gated Inwardly Rectifying K(+) (GIRK) channels. Little is known about the identity of functionally important interactions between Gbetagamma and GIRK channels. We tested the effects of all mammalian Gbeta subunits on channel activity and showed that whereas Gbeta1-4 subunits activate heteromeric GIRK channels independently of receptor activation, Gbeta5 does not. Gbeta1 and Gbeta5 both bind the N and C termini of the GIRK1 and GIRK4 channel subunits. Chimeric analysis between the Gbeta1 and Gbeta5 proteins revealed a 90-amino acid stretch that spans blades two and three of the seven-propeller structure and is required for channel activation. Within this region, eight non-conserved amino acids were critical for the activity of Gbeta1, as mutation of each residue to its counterpart in Gbeta5 significantly reduced the ability of Gbeta1 to stimulate channel activity. In particular, mutation of residues Ser-67 and Thr-128 to the corresponding Gbeta5 residues completely abolished Gbeta1 stimulation of GIRK channel activity. Mapping these functionally important residues on the three-dimensional structure of Gbeta1 shows that Ser-67, Ser-98, and Thr-128 are the only surface accessible residues. Galpha(i)1 interacts with Ser-98 but not with Ser-67 and Thr-128 in the heterotrimeric Galphabetagamma structure. Further characterization of the three mutant proteins showed that they fold properly and interact with Ggamma2. Of the three identified functionally important residues, the Ser-67 and Thr-128 Gbeta mutants significantly inhibited basal currents of a channel point mutant that displays Gbetagamma-mediated basal but not agonist-induced currents. Our findings indicate that the presence of Gbeta residues that do not interact with Galpha are involved in Gbetagamma interactions in the absence of agonist stimulation.     

Selective coupling of G protein beta gamma complexes to inhibition of Ca2+ channels

Several mechanisms couple heterotrimeric guanine nucleotide-binding proteins (G proteins) to cellular effectors. Although alpha subunits of G proteins (Galpha) were the first recognized mediators of receptor-effector coupling, Gbetagamma regulation of effectors is now well known. Five Gbeta and 12 Ggamma subunit genes have been identified, suggesting through their diversity that specific subunits couple selectively to effectors. The molecular determinants of Gbetagamma-effector coupling, however, are not well understood, and most studies of G protein-effector coupling do not support selectivity of Gbetagamma action. To explore this issue further, we have introduced recombinant Gbetagamma complexes into avian sensory neurons and measured the inhibition of Ca(2+) currents mediated by an endogenous phospholipase Cbeta- (PLCbeta) and protein kinase C-dependent pathway. Activities of Gbetagamma in the native cells were compared with enzyme assays performed in vitro. We report a surprising selective activation of the PLCbeta pathway by Gbetagamma complexes containing beta(1) subunits, whereas beta(2)-containing complexes produced no activation. In contrast, when assayed in vitro, PLCbeta and type II adenylyl cyclase did not discriminate among these same Gbetagamma complexes, suggesting the possibility that additional cellular determinants confer specificity in vivo.     

Signaling by a non-dissociated complex of G protein βγ and α subunits stimulated by a receptor-independent activator of G protein signaling, AGS8

Accumulating evidence suggests that heterotrimeric G protein activation may not require G protein subunit dissociation. Results presented here provide evidence for a subunit dissociation-independent mechanism for G protein activation by a receptor-independent activator of G protein signaling, AGS8. AGS8 is a member of the AGS group III family of AGS proteins thought to activate G protein signaling primarily through interactions with Gbetagamma subunits. Results are presented demonstrating that AGS8 binds to the effector and alpha subunit binding "hot spot" on Gbetagamma yet does not interfere with Galpha subunit binding to Gbetagamma or phospholipase C beta2 activation. AGS8 stimulates activation of phospholipase C beta2 by heterotrimeric Galphabetagamma and forms a quaternary complex with Galpha(i1), Gbeta(1)gamma(2), and phospholipase C beta2. AGS8 rescued phospholipase C beta binding and regulation by an inactive beta subunit with a mutation in the hot spot (beta(1)(W99A)gamma(2)) that normally prevents binding and activation of phospholipase C beta2. This demonstrates that, in the presence of AGS8, the hot spot is not used for Gbetagamma interactions with phospholipase C beta2. Mutation of an alternate binding site for phospholipase C beta2 in the amino-terminal coiled-coil region of Gbetagamma prevented AGS8-dependent phospholipase C binding and activation. These data implicate a mechanism for AGS8, and potentially other Gbetagamma binding proteins, for directing Gbetagamma signaling through alternative effector activation sites on Gbetagamma in the absence of subunit dissociation.     

Activation of phospholipase C-epsilon by heterotrimeric G protein betagamma-subunits

PLC-epsilon was identified recently as a phosphoinositide-hydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25- and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and alpha-subunits (Galpha(12)) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-epsilon revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of Gbetagamma subunits with the PH domains of other proteins led us to examine the capacity of Gbetagamma to activate PLC-epsilon. Co-expression of Gbeta(1)gamma(2) with PLC-epsilon in COS-7 cells resulted in marked stimulation of phospholipase C activity. Gbeta(2) and Gbeta(4) in combination with Ggamma(1), Ggamma(2), Ggamma(3), or Ggamma(13) also activated PLC-epsilon to levels similar to those observed with Gbeta(1)-containing dimers of these Ggamma-subunits. Gbeta(3) in combination with the same Ggamma-subunits was less active, and Gbeta(5)-containing dimers were essentially inactive. Gbetagamma-promoted activation of PLC-epsilon was blocked by cotransfection with either of two Gbetagamma-interacting proteins, Galpha(i1) or the carboxyl terminus of G protein receptor kinase 2. Pharmacological inhibition of PI3-kinase-gamma had no effect on Gbeta(1)gamma(2)-promoted activation of PLC-epsilon. Similarly, activation of Ras in the action of Gbetagamma is unlikely, because a mutation in the second RA domain of PLC-epsilon that blocks Ras activation of PLC failed to alter the stimulatory activity of Gbeta(1)gamma(2). Taken together, these results reveal the presence of additional functional domains in PLC-epsilon and add a new level of complexity in the regulation of this novel enzyme by heterotrimeric G proteins.     

Directly from Galpha to protein kinase A: the kelch repeat protein bypass of adenylate cyclase

One major class of G proteins typically functions as heterotrimeric complexes consisting of Galpha, Gbeta and Ggamma subunits. However, recent work in yeast has identified an atypical Galpha protein, Gpa2p, which functions without cognate Gbetagamma subunits. Two novel kelch repeat protein binding partners of Gpa2p, Krh1p and Krh2p, do not function as alternative Gbeta subunits, as initially thought, but rather as Gpa2p effectors. They directly link Gpa2p to protein kinase A, thus forming an adenylate cyclase bypass pathway that enables inputs other than cellular cAMP concentration to affect protein kinase A activity. Because mammalian protein kinase A expressed in yeast is also subject to control by the same bypass pathway, it is exciting to postulate that a functionally similar mechanism might exist in mammalian cells, and that other Galpha proteins could exhibit similar characteristics to Gpa2p.     

RACK1 binds to a signal transfer region of G betagamma and inhibits phospholipase C beta2 activation

Receptor for Activated C Kinase 1 (RACK1), a novel G betagamma-interacting protein, selectively inhibits the activation of a subclass of G betagamma effectors such as phospholipase C beta2 (PLCbeta2) and adenylyl cyclase II by direct binding to G betagamma (Chen, S., Dell, E. J., Lin, F., Sai, J., and Hamm, H. E. (2004) J. Biol. Chem. 279, 17861-17868). Here we have mapped the RACK1 binding sites on G betagamma. We found that RACK1 interacts with several different G betagamma isoforms, including G beta1gamma1, Gbeta1gamma2, and Gbeta5gamma2, with similar affinities, suggesting that the conserved residues between G beta1 and G beta5 may be involved in their binding to RACK1. We have confirmed this hypothesis and shown that several synthetic peptides corresponding to the conserved residues can inhibit the RACK1/G betagamma interaction as monitored by fluorescence spectroscopy. Interestingly, these peptides are located at one side of G beta1 and have little overlap with the G alpha subunit binding interface. Additional experiments indicate that the G betagamma contact residues for RACK1, in particular the positively charged amino acids within residues 44-54 of G beta1, are also involved in the interaction with PLCbeta2 and play a critical role in G betagamma-mediated PLCbeta2 activation. These data thus demonstrate that RACK1 can regulate the activity of a G betagamma effector by competing for its binding to the signal transfer region of G betagamma.     

Gbeta 5gamma 2 is a highly selective activator of phospholipid-dependent enzymes

In this study, Gbeta specificity in the regulation of Gbetagamma-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase Cbeta (PLCbeta) isozymes was examined. Recombinant mammalian Gbeta(1-3)gamma(2) complexes purified from Sf9 membranes stimulated PI3Kgamma lipid kinase activity with similar potency (10-30 nm) and efficacy, whereas transducin Gbetagamma was less potent. Functionally active Gbeta(5)gamma(2) dimers were purified from Sf9 cell membranes following coexpression of Gbeta(5) and Ggamma(2-His). This preparation as well as Gbeta(1)gamma(2-His) supported pertussis toxin-mediated ADP-ribosylation of Galpha(i1). Gbeta(1)gamma(2-His) stimulated PI3Kgamma lipid and protein kinase activities at nanomolar concentrations, whereas Gbeta(5)gamma(2-His) had no effect. Accordingly, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), significantly stimulated the lipid kinase activity of PI3Kbeta in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLCbeta(2) and PLCbeta(1). However, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), activated PLCbeta(3). Experimental evidence suggests that the mechanism of Gbeta(5)-dependent effector selectivity may differ between PI3K and PLCbeta. In conclusion, these data indicate that Gbeta subunits are able to discriminate among effectors independently of Galpha due to selective protein-protein interaction.     

RACK1 regulates directional cell migration by acting on G betagamma at the interface with its effectors PLC beta and PI3K gamma

Migration of cells up the chemoattractant gradients is mediated by the binding of chemoattractants to G protein-coupled receptors and activation of a network of coordinated excitatory and inhibitory signals. Although the excitatory process has been well studied, the molecular nature of the inhibitory signals remains largely elusive. Here we report that the receptor for activated C kinase 1 (RACK1), a novel binding protein of heterotrimeric G protein betagamma (Gbetagamma) subunits, acts as a negative regulator of directed cell migration. After chemoattractant-induced polarization of Jurkat and neutrophil-like differentiated HL60 (dHL60) cells, RACK1 interacts with Gbetagamma and is recruited to the leading edge. Down-regulation of RACK1 dramatically enhances chemotaxis of cells, whereas overexpression of RACK1 or a fragment of RACK1 that retains Gbetagamma-binding capacity inhibits cell migration. Further studies reveal that RACK1 does not modulate cell migration through binding to other known interacting proteins such as PKCbeta and Src. Rather, RACK1 selectively inhibits Gbetagamma-stimulated phosphatidylinositol 3-kinase gamma (PI3Kgamma) and phospholipase C (PLC) beta activity, due to the competitive binding of RACK1, PI3Kgamma, and PLCbeta to Gbetagamma. Taken together, these findings provide a novel mechanism of regulating cell migration, i.e., RACK1-mediated interference with Gbetagamma-dependent activation of key effectors critical for chemotaxis.     

RACK1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology domains in vitro.

The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the beta-adrenergic receptor kinase (betaARK), was found to bind to various phospholipids as well as the beta subunit of heterotrimeric G proteins (Gbeta) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to Gbeta (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of beta-spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1, 2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP(3)) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.     

Spatial and temporal regulation of RACK1 function and N-methyl-D-aspartate receptor activity through WD40 motif-mediated dimerization

Efficient signaling requires accurate spatial and temporal compartmentalization of proteins. RACK1 is a scaffolding protein that fulfils this role through interaction of binding partners with one of its seven WD40 domains. We recently identified the kinase Fyn and the NR2B subunit of the N-methyl-D-Aspartate receptor (NMDAR) as binding partners of RACK1. Scaffolding of Fyn near its substrate NR2B by RACK1 inhibits Fyn phosphorylation of NR2B and thereby negatively regulates channel function. We found that Fyn and NR2B share the same binding site on RACK1; however, their binding to RACK1 is not mutually exclusive (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). We therefore tested the hypothesis that RACK1 forms a homodimer that allows the simultaneous binding of Fyn and NR2B. We found that RACK1 binds to itself both in vitro and in the brain. Deletion analyses identified a RACK1-RACK1 dimer-binding site within the 4th WD40 repeat, and application of the 4th WD40 repeat or a peptide derivative to hippocampal slices inhibited NMDAR activity. We further found that in hippocampal slices, both RACK1 and NR2B associated with another WD40 protein, the beta-subunit of G protein (Gbeta), previously shown to heterodimerize with RACK1 in vitro (Dell, E. J., Connor, J., Chen, S., Stebbins, E. G., Skiba, N. P., Mochly-Rosen, D., and Hamm, H. E. (2002) J. Biol. Chem. 277, 49888-49895). However, activation of the pituitary adenylate cyclase polypeptide (1-38) G protein-coupled receptor, previously found to induce the dissociation of RACK1 from the NMDAR complex (Yaka, R., He, D. Y., Phamluong, K., and Ron, D. (2003) J. Biol. Chem. 278, 9630-9638), attenuated the association of Gbeta with RACK1 and NR2B. Based on these results, we propose that WD40-mediated homo- and heterodimerization of RACK1 mediate the formation of a transient signaling complex that includes the NMDAR, a G protein and Fyn.     

Dopamine receptor-interacting protein 78 acts as a molecular chaperone for Ggamma subunits before assembly with Gbeta

Heterotrimeric G proteins play a central role in intracellular communication mediated by extracellular signals, and both Galpha and Gbetagamma subunits regulate effectors downstream of activated receptors. The particular constituents of the G protein heterotrimer affect both specificity and efficiency of signal transduction. However, little is known about mechanistic aspects of G protein assembly in the cell that would certainly contribute to formation of heterotrimers of specific composition. It was recently shown that phosducin-like protein (PhLP) modulated both Gbetagamma expression and subsequent signaling by chaperoning nascent Gbeta and facilitating heterodimer formation with Ggamma subunits (Lukov, G. L., Hu, T., McLaughlin, J. N., Hamm, H. E., and Willardson, B. M. (2005) EMBO J. 24, 1965-1975; Humrich, J., Bermel, C., Bunemann, M., Harmark, L., Frost, R., Quitterer, U., and Lohse, M. J. (2005) J. Biol. Chem. 280, 20042-20050). Here we demonstrate using a variety of techniques that DRiP78, an endoplasmic reticulum resident protein known to regulate the trafficking of several seven transmembrane receptors, interacts specifically with the Ggamma subunit but not Gbeta or Galpha subunits. Furthermore, we demonstrate that DRiP78 and the Gbeta subunit can compete for the Ggamma subunit. DRiP78 also protects Ggamma from degradation until a stable partner such as Gbeta is provided. Furthermore, DRiP78 interaction may represent a mechanism for assembly of specific Gbetagamma heterodimers, as selectivity was observed among Ggamma isoforms for interaction with DRiP78 depending on the presence of particular Gbeta subunits. Interestingly, we could detect an interaction between DRiP78 and PhLP, suggesting a role of DRiP78 in the assembly of Gbetagamma by linking Ggamma to PhLP.Gbeta complexes. Our results, therefore, suggest a role of DRiP78 as a chaperone in the assembly of Gbetagamma subunits of the G protein.     

植物RACK1蛋白研究进展

RACK1（蛋白激酶C受体）是一种色氨酸-天门冬氨酸域（WD40结构）重复蛋白。它是一种多功能支架蛋白, 结合来自不同转导通路的信号分子并在多种哺乳动物发育过程中起关键作用。在植物中也存在RACK1同源基因, 如拟南芥基因组有3个编码RACK1蛋白质的基因, 这3个蛋白质与哺乳动物RACK1在氨基酸水平的相似性都超过75%。此外, 植物RACK1蛋白质包含的WD40数量、位置和蛋白激酶C结合位点的结构域在很大程度上是保守的。该文对植物RACK1蛋白的发现、结构及其在信号转导方面的功能进行综述。